It is proposed to continue a study of the mechanisms of 3 pyridine nucleotide-disulfide oxidoreductases: lipoamide dhydrogenase, glutathione reductase and thioredoxin reductase. Each of these flavoproteins contain an oxidation-reduction active cystine residue. In lipoamide dehydrogenase and glutathione reductase the half cystine nearer the amino-terminus of the protein interacts with the disulfide/dithiol substrate while the half cystine nearer the carbosyl-terminus interacts with the FAD. There is a base in the active center which is protonated upon 2-electron reduction forming an ion pair with thiolate, analogous to those observed in papain, glyceraldehyde-3-phosphate dehydrogenase et al. The general prinicipal of activation of thiols by ion pair formation will be explored in other enzymes. Chemical modification of the base and other active center residues will be attempted. These studies will be extended to thioredoxin reductase. The steady state and rapid reaction kinetics of thioredoxin reductase will be reinvestigated in an effort to assess the relative importance of EH2 and EH4 in catalysis. The charge transfer absorption characteristic of 2-electron reduced lipoamide dehydrogenase and glutathione reductase is pH dependent, and at low pH the charge transfer complex is not the only species present. Certain properties of the other species have been characterized in the Escherichia coli lipoamide dehydrogenase and these studies will be extended to pig heart lipoamide dehydrogenase and yeast glutathione reductase, by studying their absorbance and fluorescence properties as a function of pH. The dithiol/disulfide exchange between dihydrolipoamide and the enzyme will be studied using substrate analogs. Certain features of the gross structure of lipoamide dehydrogenase will be compared with the emerging structure of human erythrocyte glutathione reductase (sequence and X-ray structure in progress elsewhere), e.g., is the active center disulfide in the same portion of the polypeptide chain? The spectral properties of human erythrocyte glutathione reductase will be examined in reductive titrations.